Method for HLA-DR typing of total human lymphocyte sample

ABSTRACT

An HLA-DR typing test based on lymphocytotoxicity in which a vital dye-labeled total human lymphocyte sample, such as a sample of peripheral blood lymphocytes, is incubated with HLA-DR antisera, a monoclonal antibody against T cells, and complement and the DR type is determined based upon the resultant cytotoxicity as measured by the fluorescence of B cells surviving the incubation.

DESCRIPTION

1. Technical Field

The invention is in the field of immunology. More particularly itrelates to an improved HLA-DR typing test.

2. Background Art

The major histocompatability complex (MHC) of humans is a cluster ofgenes occupying a region located on the sixth chromosome. This complex,denoted HLA (Human Leukocyte Antigen), is currently divided into fivemajor gene loci, which according to World Health Organizationnomenclature are designated HLA-A, HLA-B, HLA-C, HLA-D, and HLA-DR. Theproducts of the HLA genes are commonly called "antigens". The genes ofthe A, B, and C loci encode the classical transplantation antigenswhereas the genes of the D and DR loci most probably encode antigensthat control immune responsiveness. HLA antigens are present in themembranes of human cells. Some are present in most cells of the bodywhereas others are present only in specific kinds of cells. Forinstance, HLA-DR antigens have been identified in B cells but not inresting T cells.

The HLA antigens are categorized into types that vary from individual toindividual. HLA typing is used in paternity determinations, transplantand transfusion compatability testing, blood component therapy,anthropological studies, and in disease association correlation todiagnose diseases or to predict susceptibility to disease. CurrentHLA-DR typing techniques consist of two basic methods (Joint Report from8th International Histocompatibility Testing Workshop, 1980, LosAngeles). One involves separating B cells from a total lymphocytesample, e.g. peripheral blood lymphocytes (PBL), treating the B cellswith anti-DR sera and complement, and reading the resultant cytotoxicityas an index of reactivity. The B cells are separated from the totallymphocyte population because DR antigens are present only in B cellsand B cells constitute only a small proportion, typically 10% to 25%, ofPBL. Cytotoxicity of such a small proportion of cells would be difficultto discern accurately. Numerous methods have been used previously toseparate B cells from PBL. The most common method takes advantage of thereaction of T cells with sheep erythrocytes (SRBC) to form rosettes thatcan be centrifuged through a layer of Ficoll-Hypaque, leaving the Bcells at the top of the gradient. Other methods take advantage of theaffinity of B cells for various materials such as nylon wool, Degalonbeads, and anti-human F(ab')₂ reagent. These methods suffer from variouscombinations of being time consuming or technically difficult, yieldingimpure preparations (i.e. contamination with non-B lymphocytes),providing poor absolute yields of testable B cells or, yieldingseparated B cells that have poor viability.

Monoclonal antibodies against HLA-DR antigens have been used to separateB cells from PBL for use in HLA-DR typing tests (de Krester, et al,Tissue Antigens (1980) 16:317-325).

The second basic HLA-DR typing method is the two color fluorescencetechnique (Van Rood, J. J., et al, Nature (1976) 262:795). In thismethod, to label B cells with an immunofluorescent marker, a PBLpreparation is incubated with a fluorochrome labeled anti-human Ig,washed, and then dispensed in tissue typing trays. Following sequentialincubations with DR antisera and complement the test results are read bydetermining the percent of viable B cells remaining by adding afluorescent vital dye and measuring percent viability only of thosecells having ring immunofluorescence. Although this method avoids a Bcell separation step, it requires that the cells be stained withanti-human Ig. It also is practical only when read under high powermicroscopy and, therefore, has a more demanding reading step than the Bcell separation method.

Monoclonal antibodies that bind specifically to T cells are known(Royston, et al, J Immunol (1980) 125:725; European patent applicationNo. 80300829.1 (publication number 0 017 381); and European patentapplication No. 80301357.2 (publication No. 0 018 795).

A principal object of the present invention is to provide a simple andeffective HLA-DR typing technique that (1) does not involve a B cellseparation step or a lymphocyte staining step and (2) is based oncytotoxicity function such that the sera and complement used inavailable lymphocytotoxicity tests may be used in the invention method.

DISCLOSURE OF THE INVENTION

One aspect of the invention is a B cell antigen typing methodcomprising:

(a) incubating a total human lymphocyte sample with

(i) an antibody against the B cell antigen that activates complement;

(ii) an anti-human T cell antibody that activates complement;

(iii) complement; and

(b) determining whether the B cell antigen is present in the B cells inthe sample by the resultant cytotoxicity.

Another aspect of the invention is a test kit for carrying out the abovedescribed method comprising in association:

(a) an antibody against the B cell antigen that activates complement;

(b) an anti-human T cell antibody that activates complement; and

(c) complement.

MODES FOR CARRYING OUT THE INVENTION

The invention method may be used to type any antigen that is present onB cells but not T cells. Antigens that are present exclusively on Bcells are referred to herein as "B cell antigens". While the mostprevalent use of the method will be to type HLA-DR antigens, it may beused to determine the type of other B cell antigens such as B celldifferentiation antigens. For convenience, the following description ofthe modes for carrying out the invention concerns the embodiment that isused to type HLA-DR antigens.

The typing method of the invention is based on lymphocytotoxicity.Although various human tissues may be used to obtain a total lymphocytesample (i.e. a population of T cells and B cells that has not beenfractionated to separate B cells) for the typing test, peripheral bloodis the most convenient source. At least about five ml of peripheralblood is needed for the test. Lymphocytes may be separated from theblood by density gradient centrifugation. This involves layering theblood over a gradient medium of appropriate specific gravity. The mostsuitable gradient media for separating lymphocytes from red cells andgranulocytes are high molecular weight polymers such as Ficoll (across-linked epichlorohydrin-sucrose polymer), Ficoll-Hypaque, andLymphoprep. The layered blood is centrifuged using a low centrifugalfield that avoids disrupting the cell membranes, usually 300 ×g for 20to 30 min. The lymphocytes layer on top of the gradient medium and thered cells and granulocytes pass through. Platelets are removed either bydefibrinating the blood before density gradient centrifugation or bydifferential centrifugation. Preferably the blood is defibrinatedbeforehand and the cells are suspended in a balanced salt solution at acell:solution volume ratio of 1:1 for centrifuging. Following gradientcentrifugation the lymphocytes are harvested from the interface andwashed with balanced salt solution to remove residual gradient medium.The lymphocytes may then be suspended in a growth medium, usually at3×10⁶ to 6×10⁶ cells/ml, pending use. The cells may be stored more orless indefinitely by freezing in liquid nitrogen.

The total lymphocyte sample is incubated with three reagents: (1)anti-DR sera; (2) anti-T cell antibody; and (3) complement. The anti-DRsera may be obtained from human hosts that have been immunized withHLA-DR antigen. Anti-HLA-DR antibodies are induced in human hosts bypregnancy, blood transfusion, or tissue grafting. The most useful sourceof HLA-DR typing sera is women immunized by pregnancy, since they willhave been exposed to a limited number of foreign HLA-DR antigens. HLA-DRantisera are available in predispensed typing trays from various sourcessuch as the National Institute of Allergy and Infectious Diseases. Thetyping method will preferably be carried out using a panel of such serathat are each substantially monospecific for a particular DR antigentype.

The anti-T cell antibody reagent used in the typing is one that binds toall T cells, i.e. both regulatory and effector T cells, rather than to aparticular subpopulation of T cells. It must not bind to B cells andmust activate complement rapidly and extensively. Such antibodies arecommonly called pan T cell antibodies. Antibodies of the IgM class andmost subclasses of IgG antibodies activate complement when they bind toantigens. The anti-T cell antibody is preferably of the IgM classbecause IgMs are strong complement activators. The reagent may becomposed of an antibody that binds specifically to T cells in general ora mixture of antibodies that individually bind to particular T cellsubpopulations but combined bind to the entire T cell population.

The anti-T cell antibody reagent is preferably a monoclonal antibody ofthe IgM class that binds to T cells but not to other lymphocytes. Suchmonoclonal antibodies may be produced via the somatic cell hybridizationtechnique (Kohler and Milstein, Nature (1975) 256:495-7) using animmunization protocol biased toward generating cytotoxic IgM antibodies.The antibody-producing fusion partner is prepared by immunizing a host,preferably mice, with human PBL. The PBL may be isolated from wholeblood as described above. About 40×10⁶ cells are used for theimmunization. Preferably lymphoid cells are obtained from the inguinaland axillary lymph nodes of the host during the primary immune responseand presumably before significant suppressor cell activity or somaticcell mutations of V_(H) genes can occur. Harvest will usually be doneabout 3 days post-immunization. This early harvest maximizes thelikelihood of obtaining IgM-producing lymphoid cells. The lymphoid cellsare hybridized (fused) with an appropriate myeloma cell line using afusogen such as polyethylene glycol having a Mw of about 1000 to 6000daltons. A myeloma cell line that is sensitive to a selective mediumsuch as HAT medium (Littlefield, Science (1969) 145:709-710), that fusesefficiently, and that will support stable, high level expression andsecretion of antibody by its hybridization partner is used. Whilemyeloma cells from any species may be used, murine myeloma lines havingthese characteristics are available currently and are preferred.Examples of such lines are those derived from the original MOPC-21 andMPC-11 mouse tumors that are available from the Salk Institute CellDistribution Center, PO Box 1809, San Diego, Calif. 92112.Lymphoid:myeloma cell ratios of about 0.2:1 to about 10:1 may be used,with a ratio of about 5:1 being preferred. The individual cellconcentrations will typically be in the range of 10⁶ to 10⁸, preferably1×10⁷ to 5×10⁷ cells/ml fusion medium. Balanced salt solutionscontaining 30% to 60% (w/v), preferably about 50% (w/v) fusogen, may beused as a fusion medium.

After the fusion the cells are washed with fusogen-free medium to removefusogen. They are then seeded and cultivated in the selective medium(HAT) to eliminate unhybridized parent cells and leave only hybrids thatare resistant to the selective medium and possess the immortality of themyeloma parent. The cultivation will normally take at least about 10 to14 days. Surviving hybridomas may be examined for production of anti-Tcell antibody by a standard microlymphocytotoxicity assay using T celland B cell targets (Lizak, G. E., Grumet, F. C., Human Immunology (1980)1:87). Positive hybridomas may be cloned by limiting dilution techniquesand grown in vitro or in vivo by known procedures. Monoclonal antibodyproduced by the hybridoma clones may be harvested from the culturemedium or ascites fluid by known procedures such as ammonium sulfateprecipitation, DEAE cellulose chromatography, or affinitychromatography. Further purification of the antibody, if desired, may beachieved by ultracentrifugation and microfiltration.

Rabbit serum is typically used as the complement reagent in HLA typingtests based on lymphocytotoxicity. It is believed to contain sublyticantihuman antibody which helps produce cell death. It is availablecommercially from Buxted, Pel-Freez, and others. Complement from otheranimal sources may be used provided that it produces the desired degreeof cytolysis.

The incubation of the PBL with the DR antisera, anti-T cell antibodypreparation, and complement is carried out under conditions that permitbinding between the antibodies and complement activity and will supportcells not affected by the incubation. Multi-well typing trays areavailable for containing the incubation mixture. A conventional cellgrowth medium containing serum will normally be used as an incubationmedium. The pH of the medium is physiological (pH 7.2 to 7.4). Theantibody reagents will typically be used in equivolume amounts relativeto the PBL, whereas the complement is normally used in a substantialvolume excess, e.g. at least about 5:1 to 10:1, relative to the PBLsample. A preferred protocol is to first incubate the DR antisera withthe PBL and allow the antisera to bind with any HLA-DR antigens presentin the B cells in the PBL sample. The anti-T cell antibody is then addedand the mixture is incubated to allow it to bind to the T cells in thePBL. The temperature at which the antisera and anti-T cell antibodyincubations are carried out is preferably about 37° C. The complement isthen added and the mixture in incubated, preferably at about 22° C., toallow the complement to be fixed by the antigen-antibody complexes. Suchfixation will result in cytolysis of all the T cells in the test mixtureand those B cells with which the DR antisera have reacted. In instanceswhere a DR antiserum does not react with the B cell antigens of thesample there will be a readily detectable number of residual viable Bcells.

Cytolysis may be detected and measured by loss of a vital dye, e.g.intracellular fluorescein or carboxyfluorescein, retained by livingcells, or leakage of ⁵¹ Cr or other labels from injured cells.Comparison of the test samples with negative controls permits anaccurate determination of whether the PBL sample contains a particularHLA-DR antigen. A preferred cytotoxicity determination procedure usesthe vital dye carboxyfluorescein diacetate as a label that isadministered to the PBL before the incubation. This dye is taken up,activated, and concentrated by living cells. Cytolysis permits the dyeto diffuse from the lysed cells, thereby causing lysed cells to lackfluorescence. Fluorescence in this preferred cytotoxicity determinationprocedure may be read with a standard fluorescent microscope. The livecells that survive the incubation fluoresce green. The dead cells areindistinguishable in the dark background. Readings may be made visuallyor by automated technology that detects and quantifies residualintracellular fluorescence.

The following examples further describe the invention. These examplesare not intended to limit the invention in any manner. The materials andmethods used were as follows.

Preparation of PBL Samples

Peripheral blood was drawn from healthy volunteers into acid citratedextrose (ACD) tubes and divided into aliquots. Some aliquots were heldfor three days at room temperature before being processed further whileothers were processed within 24 hr after collection. Peripheral bloodlymphocytes were isolated from the aliquots by density gradientcentrifugation of defibrinated peripheral blood samples usingFicoll-Hypaque gradient medium at room temperature, 300×g. The PBL wasplaced in Beckman tubes and cryopreserved in liquid nitrogen pendinguse. The freezing medium consisted of 3% bovine serum albumin in Hank'sbalanced salt solution, 20% glucose in RPMI 1640, and 10% dimethylsulfoxide.

B Cell Separation for Comparison Tests

Separation of B cells from fresh PBLs prepared as above was performedeither by SRBC rosetting (Gmelig-Meyling, F., Ballieux, R. E., Vox Sang(1977) 33:5-8) or on Degalon bead columns (Milford, E. L., et al, HumanImmunology (1980) 1:274). Enriched B cell populations were resuspendedin 95% fetal calf serum and 5% dimethyl sulfoxide for cryopreservationin liquid nitrogen.

DR Antisera

Oiled Terasaki trays preloaded with DR antisera defining the antigensDR1 through DRw10 at one μ1/well were used.

Anti-T Cell Monoclonal Antibody

For immunization, 4×10⁷ PBL obtained from a blood donor (HLA type A2,A28; B27, Bw44; Cw4, Cw2) were suspended in phosphate buffered saline(PBS) and administered into each footpad of a 6-week old BALB/c femalemouse. Three days post-immunization the inguinal and axillary lymphnodes and the spleen were harvested, and the lymphoid cells weresuspended in Dulbecco's Modified Eagle's Medium (DMEM). For fusion thelymphoid cells were mixed at a ratio of 5:1 with SP2/08A2 mouse myelomacells, centrifuged at 400×g for 10 minutes at 22° C., and the pelletresuspended in 1 ml of 50% polyethylene glycol (mw1500) in DMEM. Thismixture was gradually resuspended into 7 ml DMEM over a six-minuteperiod at room temperature, centrifuged, and ultimately resuspended inDMEM supplemented with 15% newborn calf serum (NCS) in a finalconcentration of 10 million viable cells per cc. One-tenth cc of thiscell suspension was then placed into each of 60 wells of a 96 welltissue culture plate. Following overnight incubation at 37° C. in 6%CO₂, each well received an equal volume of 15% NCS in DMEM supplementedwith 8.0×10⁻⁵ M hypoxanthine, 2×10⁻⁷ M aminopterin and 1.3×10⁻⁵ Mthymidine (HAT) for selection of fused cells. On each of the followingthree days, half of the volume of each well was replaced with fresh HATmedium. By day 10 those wells showing continued growth in the HAT mediumhad supernatant samples drawn off to test for antibody activity by astandard microlymphocytotoxicity assay using T cell and B cell targets.One parent well contained a clone, subsequently named TM1, whichexhibited specificity for T cells but not B cells or monocytes. It isbelieved that TM1 antibody binds to the red blood cell rosette receptor.This clone was selected and subcloned in vitro by the method of limitingdilution. Syngeneic mice, pretreated with Pristane intraperitoneally(ip), were given approximately 10 to 30×10⁶ of the cloned TM1 cells ipand the resultant ascites fluid was harvestd over the next several weeksfor use directly in the invention DR typing method.

Complement

Eight week old rabbit serum (Pel-Freez, Rogers, Arizona) locallyprescreened for cytotoxic activity against B and T lymphocytes was used.

Invention Typing Method

Frozen PBL samples were thawed rapidly in a 37° C. water bath. When onlya small crystal of ice remained the Beckman tube was filled slowly with20% heat-inactivated fetal calf serum (HIFCS) in RPMI. The cellsuspension was transferred to a 1.5 cc tube and the tube was filled with20% HIFCS/RPMI and centrifuged for 3 min at room temperature, 300×g. Thesupernatant was removed and discarded. A working solution ofcarboxyfluorescein diacetate (c-FDA) was made by diluting a stockacetone solution of c-FDA (10 mg/ml) in phosphate buffered saline (PBS)at 1:250. The cell pellet was resuspended gently in 150 μ1 of the c-FDAworking solution and the suspension was incubated at 37° C. for 15 minin the dark. Following the incubation one ml of 20% HIFCS/RPMI was addedand the suspension was tranferred to a 12×75 mm tube.

The cell suspension was then underlayered with one ml of 10%Ficoll-Hypaque and centrifuged at 350×g for 6 min at 20° C. The cellinterface was removed and diluted to approximately 1.4 ml with 20%HIFCS/RPMI, and the suspension was centrifuged for 3 min at roomtemperature, 300×g. The supernatant was removed and the cell pellet wasresuspended in 20% HIFCS/RPMI to approximately 5000 cells/μ1 (about 100μ1 of 20% HIFCS/RPMI).

The viability of cells was checked by adding one drop of 0.4% ethidiumbromide to one drop of the cell suspension. Live cells fluoresce greenand dead cells fluoresce orange under a fluorescent microscope.

One μ1 of the c-FDA labeled cell suspension was added to each test wellof the preloaded Terasaki tray and the tray was incubated for 1/2 hourat 37° C. in the dark. One μ1 of the anti-T cell monoclonal antibodypreparation diluted 1:20 in 3% bovine serum albumin in Hank's basic saltsolution was then added to the wells. Two negative control sera wereused on the tray. Monoclonal antibody was not added to one of them toprovide a control for the activity of the monoclonal antibody. The traywas then uncubated for 1/2 hr at 37° C. in the dark. Ten μ1 of thepretested rabbit complement reagent was then added to each well and thetray was incubated for 2 hr at 20° C. in the dark. The tray was thenblotted with an absorbant paper to remove oil in the wells andfacilitate readings.

A small volume from the top of each well was removed and read under a 6Xobjective on a standard fluorescent microscope using either dark fieldor epiillumination with appropriate excitation and barrier filters forfluorescein fluorescence. Dead cells are not visible since they havelost their intracellular fluorescence. Live cells will fluoresce green.The cell death (i.e., fluorescence) of the test wells are compared tothat of the negative control well to determine the DR type of the samplePBL. Cytotoxicity is scored on a scale of 1 to 8, using the negativecontrol well as a relative "100%" assigned viability standard; thus

8=85%-100% relative cell death

6=50%-85% relative cell death

4=<50% relative cell death.

Comparison Typing of B Cell Enriched Populations

Enriched B cell populations were treated with c-FDA as described aboveand diluted with 20% HIFCS/RPMI into a concentration of 2×10 per cc. Oneμ1 of the cell suspension was added to each well of DR alloantiseraloaded Terasaki trays and the trays were incubated at 37° C. for onehour. Five μ1 of complement were then added to each well and following asecond incubation at 20° C. the trays were read in the manner describedabove.

HLA-DR typing of nine individuals was carried out using the inventionmethod and the comparison typing methods. Details and results of thesetests are reported in the table below.

    __________________________________________________________________________    DR TYPING                                                                     DR Sera        Results                                                                 Other DR                                                                            CG    DN   LF   BF   BI   JB   AM   MK    TK                   Specificity                                                                            Specifi-                                                                            (DR5,w10)                                                                           (DR5,7)                                                                            (DR4,--)                                                                           (DR7,--)                                                                           (DR1,4)                                                                            (DR2,w6)                                                                           (DR2,3)                                                                            (DRw6,w8)                                                                           (DRw9,--)            and Name cities                                                                              1 S 2 3                                                                             1 S 2 3                                                                            1 2  1 3  1 3  1 2  1 3  1 3   1                    __________________________________________________________________________                                                             3                    DR1: B4289                          8 8                                            Bode                           6 8                                            Lewan                          8 8                                       DR2: JoneC                                                                             DRw6                            6 4  8 6  6                               Olsen                               6 4  8 6                                  Rinde                               6 4  6 4                                  S3066                               6 6  6 8                                  S8332                           6   6 4  8 6                             DR3: Berge                                    8 4                                  Lake                                     8 4                                  Lilli                6                   6 4                                  Pett                                                                              Drw6             4 6                 8 6                                  Tate                                     6                               DR4: Bra77                6 6       6 6                                            Murph                8 6       6 6                                            Niels              6 4         6 6                                       DR5: N1107                                                                             DRw6   6 N  6 6 6 6                       6                               Ocken                                                                             DRw6  6 8 6 4                                                                             8 8 8 6                                                                            4     4        4         6                               Stiev     8 8 6 4                                                                             8 8 6 N                                                                            6               4    4   N                               SKI74     6 8 6 4                                                                             8 8 6 6                                                       Cu30                                                                              DRw8  6 6 6 8 8 8 6                                                                            6     6             4 4  6                               JB153      6 N  6 6 8 N                                                  Drw6:                                                                              JoneC                                                                             DR2                             6 4  8 6  6                               JoneL                                                                             DR3                             6 4  8 8  6                               Ocken                                                                             DR5   6 8 6 4                                                                             8 8 8 6                                                                            4    4         4         6                               Carne                                                                             DR1,DR2           6        4 6  6 4  8 6  6 4                             Charl                           4        6    6 4                        DR7: Chadw           8 8 6 8   6                                                   L701                                                                              DR3         8 8 6 6   6                                                   JH371           6 8 8 8   6 4                                                 Me712           8 8 6 6   6                                                   Schul                                                                             DR2         8 8 6     6                                              DRw8:                                                                              Kubic                                         8 6                        DRw9 Culps                                                                             DRw10 6 8 6 6                                   8 4                       JR50       6                                        8 6                  DRw10:                                                                             Chan      N N 6 4                                                             Culps                                                                             DRw9  6 8 6 6                                   8                         NW145     6 8 6                                                          __________________________________________________________________________     Method:                                                                       1 = TM1 treated PBLs                                                          S = TM1 treated PBLs stored at room temperature for 3 days before             isolation and cryopreservation                                                2 = B cell enriched  Degalon beads                                            3 = B cell enriched  SRBCrosetting                                            Cytotoxicity scored on a standard scale of 1 to 8. For simplicity all         spaces with negative (ie "1") reactions are left                              N = Not done.                                                            

In all cases the DR typing reactions in the TM1 technique were at leastas strong and clear as the comparison method(s). It is also noteworthythat even for samples that had been stored for 3 days (before PBLisolation) TM1 typings were clear and strong. Similar 3 day storageexperiments were not tried with the comparison DR typing methods becauseextensive prior experience had shown such storage to be extremelydeleterious to typing by those methods. In addition to the serologicaldata shown in the table, additional cells covering all of thealloantigens DR1-DRw10 were typed by the TM1 method and either or bothof the comparison methods, and in every case the phenotype assignmentsby the TM1 method results were at least as good and generally better,than the comparison method.

Comparisons of post-cryopreservation yields of cells suitable for DRtyping, per 10⁶ PBLs isolated from starting whole blood, showed thefollowing ranges: (1) SRBC-rosetting, 0.5-1.0×10⁵, with 80%-95% B cellpurity (defined by reactivity with the monomorphic, monoclonal anti-DRantibody L243); (2) Degalon beads, 0.3-0.6×10⁵, with 90%-100% purity;(3) TM1, 0.5-1.0×10⁵, with 90%-100% purity. It should also be noted thatqualitatively the reactivity was judged by the technologists performingthe typing to be in the order TM1≧Degalon>SRBC-rosetting.

The test kits for carrying out the invention DR typing method willcontain as basic ingredients given amounts of each DR antiserum, theanti T-cell antibody, and complement. These ingredients will bedispensed in suitable containers. The kit will also typically include acytotoxicity marker, e.g. a vital dye, gradient medium, and directionsfor performing the method. The DR antisera may optionally be preloadedinto typing trays or an unloaded typing tray may be included in the kit.The kit components may be packaged in a conventional manner.

Modifications of the above described modes for carrying out theinvention that are obvious to those of ordinary skill in theimmunodiagnostic art or related arts are intended to be within the scopeof the following claims.

We claim:
 1. An HLA-DR typing method comprising:(a) incubating a total human lymphocyte sample with(i) an anti-HLA-DR antibody that activates complement; (ii) and anti-human T cell antibody that activates complement; and (iii) complement; and (b) determining whether the HLA-DR antigen to which the anti-HLA-DR antibody binds is present in the B cells in the sample by comparing the viable B cell content of the incubated sample with a negative control.
 2. The method of claim 1 wherein the sample is a sample of peripheral blood lymphocytes.
 3. The method of claim 1 or 2 wherein the anti-human T cell antibody is a monoclonal antibody.
 4. The method of claim 3 wherein the monoclonal antibody is of the IgM class.
 5. The method of claim 1 or 2 wherein the complement is rabbit complement.
 6. The method of claim 1 or 2 wherein the sample is labeled with a vital dye before the incubation and the resultant cytotoxicity is based on the loss of the vital dye by lysed cells.
 7. The method of claim 6 wherein the vital dye is carboxyfluorescein diacetate.
 8. The method of claim 2 wherein the anti-human T cell antibody is a monoclonal antibody of the IgM class; the complement is rabbit complement; the sample is labeled with carboxyfluorescein diacetate before the incubation and the resultant cytotoxicity is based on the loss of the label by lysed cells.
 9. The method of claim 1, 2, or 8 wherein the incubation of the sample with the HLA-DR antibody, anti-human T cell antibody, and complement is carried out in separate steps.
 10. The method of claim 9 wherein the HLA-DR antibody incubation step and the anti-human T cell antibody incubation step are each carried out at about 37° C. and the complement incubation step is carried out at about 22° C. 